Glucose-to-Resistor Transduction Integrated into a Radio-Frequency Antenna for Chip-less and Battery-less Wireless Sensing

To maximize the potential of 5G infrastructure in healthcare, simple integration of biosensors with wireless tag antennas would be beneficial. This work introduces novel glucose-to-resistor transduction, which enables simple, wireless biosensor design. The biosensor was realized on a near-field communication tag antenna, where a sensing bioanode generated electrical current and electroreduced a nonconducting antenna material into an excellent conductor. For this, a part of the antenna was replaced by a Ag nanoparticle layer oxidized to high-resistance AgCl. The bioanode was based on Au nanoparticle-wired glucose dehydrogenase (GDH). The exposure of the cathode-bioanode to glucose solution resulted in GDH-catalyzed oxidation of glucose at the bioanode with a concomitant reduction of AgCl to highly conducting Ag on the cathode. The AgCl-to-Ag conversion strongly affected the impedance of the antenna circuit, allowing wireless detection of glucose. Mimicking the final application, the proposed wireless biosensor was ultimately evaluated through the measurement of glucose in whole blood, showing good agreement with the values obtained with a commercially available glucometer. This work, for the first time, demonstrates that making a part of the antenna from the AgCl layer allows achieving simple, chip-less, and battery-less wireless sensing of enzyme-catalyzed reduction reaction.

. TEM images of (A) AgNPs and (B) AuNPs, which were used in this study.
Size distribution, zeta potential and the concentration of the prepared AgNPs and AuNPs were determined by Zetasizer Ultra (ZSU5700, Malvern Panalytical, UK). The size distribution measurements were carried out in standard polystyrene (PS) cuvettes using multi-angle dynamic light scattering measurements (MADLS) in three different angels of backscatter, side scatter and forward scatter to give a global fitting for better resolution. The data are presented in Fig. S2A.
Zeta potential measurements were performed by using Zetasizer Ultra using the disposable folded capillary cells (DTS1080). On average, zeta potentials were found to be -39 mV and -44 mV for the negatively charged citrate-stabilized Ag and Au NPs. The original data on measurements of zeta potential are presented in Fig. S2B.  Determination of the concentration, using the Zetasizer Ultra, was based on multi-angle dynamic light scattering measurements (MADLS). The technique transforms the intensity-weighted particle size distribution to the absolute concentration using the derived count rate and the optical properties of the nanoparticles and the suspension medium 4 . Before measuring the concentration of the colloidal nanoparticles, the background intensity of the dispersant (ultrapure water) was measured three times in order to subtract the background contribution. The results based on DLS measurements showed the concentration of ≈ 2 × 10 11 and ≈ 1 × 10 12 particles/ml for Ag and Au NPs, respectively (Fig. S3A). NP dispersions characterised by MADLS were also assessed by measurements of the localized surface plasmon resonance (LSPR) of the NPs using a UV-vis spectrophotometer (UV-1800 SHIMADZU, Kyoto, Japan). By assuming that the whole added amount of Ag and Au salts have been reduced and formed nanoparticles; the only attenuating products are nanoparticles, the concentration of AgNPs and AuNPs can be calculated using Beer Lambert's law 5,6 . The extinction coefficients were taken to be 3.67 × 10 8 and 1.45 × 10 10 M -1 cm -1 from the previously reported values at the wavelength of 520 nm and 406 nm for Au and Ag nanoparticles, respectively 7,8 . The concentrations of dispersions estimated from these UV-vis experiments are presented in Table S1 and the corresponding absorption plots are shown Fig.  S3B. The obtained concentrations from UV-vis measurements were found to be ≈ 5 × 10 11 and ≈ 5 × 10 12 particles/ml for Ag and Au NPs which were consistent with the one calculated by Zetasizer Ultra. 10,6 ± 0,9 Method C AgNPs/ particles/ml C AuNPs/ particles/ml MADLS 2,2 × 10 11 ± 0,6 1,3 × 10 12 ± 0,2 UV-vis 5,0 × 10 11 ± 4,1 5,7 × 10 12 ± 0,9 Method ζ-potential AgNPs /mV ζ-potential AuNPs /mV Zeta potential -38,6 ± 2,0 -44,4 ± 2,1 To probe possible particle aggregation Small-angle X-ray Scattering (SAXS) data were collected using a XEUSS 3.0 instrument from Xenocs (Grenoble, France) with a photon energy E = 8 keV, using the Cu Kα radiation (λ = 1.54 Å). The signal was collected using a Pilatus 300K detector (Dectris). All the experiments were performed in vacuum at room temperature. The samples used for the experiment were: AuNP dispersion and AgNP dispersions were in water. All the samples were filled in 80 mm long borosilicate glass capillaries (WJM-Glass, Berlin, Germany) with the outside diameter of 1.5 mm and wall thickness of 0.01 mm. Standard corrections were done using XSact software from Xenocs 9 and the fits were done with SASView 10 to the dilute sphere model 11 , so the final curves, shown in Fig. S4A, represents the result of the absolute intensity after subtraction of the solvent (water). The good agreement with the dilute sphere model and the linear behavior of the Guinier law fit at low Q values shows that there was no aggregation of nanoparticles. The elemental analysis of the Ag and Au NPs was also performed using the EDX (Energy-dispersive X-ray spectroscopy) on TEM and the results confirmed the high purity of the synthesized particles (Fig.  S4). EDX spectrum of AgNPs shows a strong signal at 3 keV which is the main characteristic peak for AgNPs (Fig. S4B). The other peaks around 22 keV and 25 keV also correspond to the binding energies of AgNPs 12,13 . For AuNPs, the spectrum indicates a strong peak at 2.2 keV (Fig. S4C). There are some characteristic peaks at 8.5, 9.8 and 11.5 keV which are also relevant to metallic AuNPs 13,14 . In EDX spectrum of both Ag and Au NPs some secondary crystals can be observed which are representative of sodium, carbon and oxygen coming from citrate groups. It should be mentioned that the copper peaks observed in both spectra correspond to the TEM grid holder.

S2. Resistance measurement of AgNPs or AgNP-AuNP layers during the electrochemical oxidation and reduction
In order to prove that AgNP or AgNP-AuNP comprised layer enables not only glucose-to-resistance transduction, but a more general redox-reaction-to-resistance transduction, the resistance of the layer made from AgNPs or AgNP-AuNP was measured during its electrochemical oxidation and reduction (Ag/AgCl conversion). For this, two potentiostats were connected to the SPE containing the transduction layer. One potentiostat was used to run cyclic voltammetry in three electrode mode with the SPE as working and external reference and counter electrodes. The applied potential ranged from -0.3 to +0.3 V and the scan rate was 10 mV s -1 . The other potentiostat was used to run chronoamperometry by applying a constant potential of 5 mV between the working and the counter electrodes on the SPE bridged by NP layer. The measured current by chronoamperometry was used to calculate the resistance of the layer in accordance to the Ohm's low. The connections of electrodes and potentiostats are illustrated in Fig. S5.

S3. Characterization of the transduction layer by different techniques: SEM and EDX
After electrooxidation of deposited NPs, AgNPs (100 %) or AgNP-AuNP mixture (80:20 %), the transduction layers were inspected by SEM. Fig. S6a and S6b show the SEM images of deposited AgNPs after electrooxidation. It is clear that the deposited layer is uneven/nonhomogeneous. As can be seen from Fig. S6b, AgNPs after electrooxidation form ≈ 300-900 nm different-shaped clumps. This is most probably due to AgNPs conversion to AgCl particles during electrooxidation. As confirmed by EDX spectra, the total amount of Cl was found to be ≈ 42 atomic percentage (Fig. S6c). Following these studies, fresh NP layer (before oxidation) was also evaluated by SEM (Fig. S6f) and the obtained results confirmed our hypothesis that the changes in morphology is caused by the electrooxidation of Ag to AgCl in PBS solution. Besides that, as shown in Fig. S6b and S6e, some small holes in the Ag and Ag-Au films are observed, which indicates that the electrochemical oxidation process leads to the formation of a porous surface with different thickness. This was confirmed by performing cross-sectional SEM images (Insets in Fig. S6f). These results also showed that the thickness of Ag-Au film deposited on SPE is ≈ 2 µm. Further, it should be noted that the AgCl particles form an overall compact structure, with some porosity. This AgCl structure bridged two electrodes on SPE as a transduction layer of relatively high resistance (as compared to the AgNPs layer).

S4. Characterisation of AgNP and AgCl containing layers by electrochemical impedance spectroscopy
In order to investigate the structure of the transduction layer resulting from Ag/AgCl conversion of AgNPs, electrochemical impedance spectroscopy (EIS) was conducted on SPE modified with AgCl-AuNPs in PBS solution at different AC voltage amplitude. The AC amplitudes ranged from 5 to 200 mV with the DC applied potential of 0.0 V. The EIS was run in the frequency range of 10 MHz to 0.1 Hz.
Interpretation of the EIS data accounted for porous structure (Fig. S7A) and were fitted with the electrochemical circuit illustrated in Fig. S7B. The EIS data can be explained by the presence of two layers on the electrode surface; the porous AgCl-AuNPs layer exposed to the electrolyte solution grown by electrochemical oxidation and the thin layer of Ag-AuNPs (Fig. S7A). Due to the surface inhomogeneity (rough and porous), the capacitive element (C dl ) does not show the ideal properties and accordingly was replaced by the constant phase element (Q) 15,16 . R s is the resistance of the electrolyte solution (PBS solution). R 1 and Q 1 are the resistance and constant phase element of the electrochemically grown AgCl-AuNPs layer exposed to the electrolyte. These characteristics depend on the defective structure or porosity of the layer 17 . On the other side, R 2 and Q 2 are the resistance and constant phase element of the layer of Ag-AuNPs deposited on the substrate.
The values of the proposed equivalent circuit obtained by fitting the experimental results shown in Fig.  S7C and S7D are summarised in Table S2. The value of Q 1 is lower if compared to Q 2 which could be related to the higher thickness of the AgCl-AuNPs layer compared to the layer of Ag-AuNPs. The porous structure of AgCl-AuNPs layer leads to the lower charge transfer resistance, measured for this layer (R 1 compared to R 2 values). It is also worth noting that by increasing the AC voltage amplitude from 5 to 200 mV, the values of charge transfer resistance for AgCl-AuNPs layer (R 1 ) decreases ( Fig. S7C and  S7D). This indicates the reduction of AgCl to Ag by the applied AC voltage, which can be modelled by the presence of a duplex layer on the substrate, as mentioned above. The present results describe a simple modelling of the AgCl to Ag conversion at low frequencies. However, association or correlation of these data to higher MHz frequencies, which are used in reading the RF antenna, is not easy to make. Though to consider possible correlations might be important for understanding RF effect on Ag/AgCl reactions.

S5. Characterisation of GDH modified electrodes
From comparison of CVs recorded with electrodes with 4-ATP immobilised GDH (Fig. 1A, main text) vs without 4-ATP immobilisation (Fig. S8A), it can be concluded that the maximum current density for 4-ATP modified electrodes is ≈ 15 times higher (0.75 mA cm -2 compared to 0.05 mA cm -2 ). This confirms an essential role of 4-ATP for GDH immobilization in facile DET contact with the electrode surface. The immobilisation follows electrochemical transformation of 4-ATP. This is shown in CV for the electrode labelled as GCE/PEI/AuNPs/4-ATP. The CV shows a reversible redox process at the potential of 0.2V (vs. SCE) which indicated the formation of the redox active specie (Fig. S8B).
According to the literature, 4-ATP can be oxidized at pH 7 to 4-mercapto-N-phenylquinone diimine (MPQD). This product is then hydrolysed rapidly to 4-mercapto-N-phenylquinone monoimine (MPQM) [18][19][20] . It has been reported that covalent attachment of enzyme on the surface of the MPQM-modified electrode occurs via a Schiff base formation between quinone groups of MPQM and the primary amino groups of glucose dehydrogenase. Since the redox potential of MPQM (≈ 0.2 V vs. SCE) is higher than the potential of the enzymatic reaction catalyzed by GDH (≈ -0.06 V vs. SCE), the MPQM acts as a crosslinking agent and not as a redox mediator. This explanation supports the statement about direct electron transfer coupling between the electrode and the GDH enzyme 18 .

S6. Repeatability of the wireless biosensor response
Ten repeated responses to 6 mM of glucose have been recorded with the same SPE bridged with AuNP-AgNP layer which was electrooxidised to AuNP-AgCl transduction layer. Each measurement of glucose reduced AgCl to Ag. To prepare the transduction layer for the repeated measurement, after each response of the biosensor to glucose, the SPE with the transduction layer was place into PBS and the layer was electrooxidised to AgCl state by applying 70 mV for 12 seconds. After that a new measurement of glucose was carried out. The response times of the wireless biosensor obtained during these repeated measurements are shown in Fig. S9.   Fig. S9. The response time to 6 mM of glucose recorded with the biosensor tag. The same SPE modified with AuNP-AgNP mixture and oxidized to kΩ level was re-used. Data are from two independently prepared SPE. The GDHbased bioanode was connected to the cathodic transduction layer as a separate AuNP/4-ATP/GDH modified electrode (coupling is shown in Fig. 1C, main text).

S7. Description of equivalent electrical circuit of the chip-less wireless biosensor
The entire wireless biosensor is comprised of RF antenna connected to an SPE which hosts GDH based bioanode and AgCl based cathode layer, i.e., the bioanode-cathode connection which enables glucoseto-resistance transduction (see Fig. 4C, main text). It is important to note that only the cathode layer is included into the RF antenna circuit (tag antenna circuit), which is wirelessly addressed by the VNA based antenna reader. To better understand features of the wireless biosensor its antenna circuit is here discussed in term of equivalent circuit. To propose equivalent circuit for the biosensor tag design, function S11, and thus, f 0 and Q were determined with the SPE, containing AgCl or Ag transduction layer, immersed in the solution of different ionic strength (data in Table S3). Table S3. Values of f 0 and Q determined for the tag, RF antenna with connected SPE (see Fig. 4C, main text). The SPE contained AgCl, or Ag comprised transduction layer specified as the material of transduction layer: AgCl or Ag. The SPE with transduction layer was immersed into the solution of different ionic strength (noted as I in mM) which was based on diluted or concentrated PBS.

Material of transduction layer
AgCl Ag Media where the SPE with transduction layer is exposed to  The characteristic frequency (f 0(AgCl) ) and (B) Q-factor (Q (AgCl) ) of the antenna with a coupled SPE exposed to air or immersed into solution of different ionic strength (I, in mM). The SPE hosts a transduction layer comprised of AgCl. Equivalent circuit representing the antenna circuit is presented in Fig. 3D, main text, and discussed in Fig. 4C, main text.
Circuit C(c) in Fig. 4C represents a suggestion of the complete biosensor tag antenna circuit. This equivalent circuit was used to derive equations (Eq. S1, S2 and S3) for rigorous mathematical fitting of the experimental f 0 and Q dependencies on ionic strength (practically estimated by R2). The mathematically modelled best fits are shown in Fig. S10A and S10B.
The analytical equations used to calculate the f 0 and Q values are presented below.
The condition for the characteristic frequency,  0 = 2f 0 , is Z im = 0. This leads to the following equation.
Eq. S2 The equation to find the bandwidth frequencies (power at Full Width Half Maximum, FWHM) can be expressed by the following equation: Eq. S3 The Eq. S3 results in a sixth-degree Cardan polynomial of the type , whose ( ) = 6 + 4 + 2 + roots can be found using radicals. From these roots, two are real positive solutions . These two ( 1 , 2 ) solutions correspond with the limits of the bandwidth, which can be used to find the Q value: The equations have been written in Mathematica symbolic language and fitted to experimental f 0 and Q values. As can be seen ( Fig. S10A and S10B) that the modelled curves relatively well describe experimentally obtained dependencies of f 0 and Q on the ionic strength of solution where the transduction layer comprised AgCl is immersed to. The values of the circuit elements derived from the fitting are presented in the main text and are as follows. For R 1 , L 1 , C 1 , and C 2 the are equal to 0.0417 , 5.61·10 -9 H, 2.62·10 -8 F, and 2.36·10 -8 F, respectively. Summarising, the fitting relatively well approximates the experimental data and, thus, confirms that the suggested equivalent circuit can be used for the representation of the antenna circuit of this novel biosensor tag. Table S4, additionally, presents a list of simple equations, which support simple interpretation of the equivalent circuit of the biosensor tag antenna circuit. Table S4. Summary of equations for estimating characteristic frequency (f 0(Ag) , f 0(AgCl) ) and Q-factor (Q (Ag) , Q (AgCl) ) of tag antenna with a coupled SPE exposed to air, immersed into H 2 O and solution of different ionic strength. The SPE hosts a transduction layer comprised of Ag or AgCl . The table lists also experimental values of f 0 and Q obtained at specified conditions. Values R 1 , R 2 , L 1 , C 1 , and C 2 are elements of the circuit shown in Fig. 3D and Fig. 4C.       S11. Chronoamperogram shows the current-time dependence obtained during oxidation of cathode layer comprised of Ag-AuNPs to AgCl. The oxidation resulted into the layer resistance of MΩ level. The oxidation to AgCl in the transduction layer was achieved at 200 mV applied voltage for 120 s in PBS solution. Included is the average charge, needed to convert Ag to AgCl, found for 11 electrodes prepared for measurements of 11 different concentrations of glucose spiked in whole blood.